The present invention relates to forensic identification of human urine samples.
Sample adulteration is a serious problem in forensic urine drug testing. Attempts to circumvent identification of drug abuse have led to the use of adulterants and substitutions in xe2x80x9curinexe2x80x9d samples provided for drug testing (see Ray H. Liu and Bruce A. Goldberger, xe2x80x9cHandbook of Workplace Drug Testingxe2x80x9d, AACC Press, 1995). For example, animal urine and non-urine liquid samples are often submitted as substitutes for human urine. For forensic purposes, it is important to positively identify human urine to the exclusion of substitute liquid samples.
Sample adulteration is usually achieved by substitution, dilution, or addition of adulterant into urine samples. The current methods for detecting sample adulteration test samples for pH, specific gravity, and creatinine concentration. However, the existence of a broad range of pH and specific gravity for urine samples limits the usefulness of these test results.
Creatinine is a normal constituent of urine (its concentration in the urine ranges from 15 to 300 mg per dl) and creatinine test is useful for identifying urine samples. However, it is difficult to use creatinine test to distinguish an animal sample from a human one because creatinine exists in both human and animal urine samples.
The present invention features a test kit and a simple, speedy and cost efficient method of identifying human urine samples. It allows for on-site identification of human urine samples without any equipment.
The test is a colloidal gold particle based Sandwich immunoassay for an antigen in human urine, e.g., human IgG, human IgA or human albumin. Because the test is specific for an antigen in human urine, it can positively identify a human urine sample to the exclusion of liquid substitutions. The sensitivity of this test also allows for the exclusion of grossly diluted human urine samples. In addition, this test can exclude human urine samples which have been so adulterated that the human antigen can no longer be detected.
Thus, this invention features a method of identifying or verifying a human urine sample with a test kit which has a chromatographic medium which contains (a) a collection of colored particles coated or otherwise attached with a first plurality of antibodies to a human antigen which exists in human urine, and (b) a first section containing a second plurality of antibodies to the same antigen. The colored particles are used as a visual marker for the test and include color and fluorescence particles known in the art, such as those described, disclosed or cited in U.S. Pat. No. 5,712,172, incorporated by reference herein in its entirety. Specifically, the colored particles include colloidal gold particles, colored latex, dye sols and carbon sols. In a preferred embodiment, colloidal gold particles are used. The antibody coated colloidal gold particles are capable of moving within the chromatographic medium by capillary action. The second plurality of antibodies is immobilized to the first section within the chromatographic medium. A test liquid sample is brought into contact with the chromatographic medium so as to move the colloidal gold particles by capillary action towards the first section. If the test liquid sample contains the antigen, the colloidal gold particles would aggregate in the first section in the form of antibody/antigen/antibody-coated colloidal gold particle Sandwiches. Such aggregation is observable by the formation of a colored band in the first section of the chromatographic medium. Therefore, the chromatographic medium is observed for the appearance of a colored band in the first section as an indication that the liquid sample is human urine.
By xe2x80x9cchromatographic mediumxe2x80x9d is meant an absorptive material (preferably solid or semisolid) which, when brought into contact with a liquid, allows the flow of a mobile liquid phase over a stationary solid or semisolid phase. In addition, it allows the mobile liquid phase to carry colloidal gold particles in its movement over the stationary solid phase. The absorptive material is optionally deposited on or affixed to a nonabsorptive solid support (e.g., a strip of plastic). The absorptive material includes, but is not limited to, paper, membrane, pad, or a combination thereof. In a preferred embodiment, the membrane is selected from materials including, but not limited to, nitrocellulose, nylon and polyvinylidene bifluoride. In another preferred embodiment, the chromatographic medium comprises a strip of absorptive materials placed on a solid support and the strip of absorptive materials comprises a pad containing the colloidal gold particles and a membrane having a section precoated with antibodies to the antigen in human urine.
Colloidal gold particles have been used in diagnostic assays (Buechler et al., Clin. Chem. 38:1678-1684 (1992); Snowen and Hommel, J. Immunol Methods 140:57-66 (1991); and Harvey et al., IVD Technology 34-40, May/June (1996)). In a preferred embodiment, gold particles with a diameter of 5-100 nm are used in this invention. In a further preferred embodiment, gold particles with a diameter of 10-50 nm are used in this invention. In an even further preferred embodiment, gold particles with a diameter of 20-40 nm are used in this invention.
The antibodies attached to the colloidal gold particles and immobilized to the chromatographic medium may be polyclonal antibodies or monoclonal antibodies.
The antibodies can be attached to the gold particles by simple absorption or other means known to those skilled in the art, e.g., soaking the gold particles in an antibody solution. A gold sol pad can be made by soaking a pad in a suspension containing protein coated gold particles and allowing the wet pad to air-dry.
The antibodies can be attached to the membrane by airbrush spray technique or ink jet printing. The membrane is then allowed to air dry to immobilize the antibodies to a designated section.
A preferred antigen is one that is not present in animal urine samples, e.g., human IgG, human IgA or human albumin. In a further preferred embodiment, human IgG is the selected antigen.
In a preferred embodiment, the chromatographic medium contains a second collection of colloidal gold particles which are attached with a second antigen. The chromatographic medium also has a second section which contains antibodies to the second antigen and these antibodies are immobilized to the medium (alternatively, the gold particles are attached with the antibodies while the antigen is immobilized to the medium). When this second collection of colloidal gold particles move to the second section, they aggregate to form a second colored band because of the interaction between the second antigen and its antibody. The absence or presence of this second colored band can be used as an indication whether the chromatographic medium is defective. Preferably, the second antigen is not present in human urine samples, e.g., rabbit immunoglobulin G.
In another preferred embodiment, the chromatographic medium comprises a sample pad, a gold sol pad, a membrane, and optionally an absorbent pad (also called a sink pad) affixed to a strip of plastic support in that order. The plastic support is selected from materials including, but not limited to, polystyrene, polyester, and polyvinyl chloride (PVC). The sample pad is affixed to the support at one end of the strip for absorbing liquid test sample while the optional absorbent pad is at the other end of the strip. The absorbent pad is made of absorptive materials, including, but not limited to, paper for absorbing liquid from the membrane and facilitating capillary movement of the liquid sample and the gold particles. The gold sol pad is placed between the sample pad and the membrane and partially overlaps with both. The gold sol pad contains the colloidal gold particles. The membrane contains a first section with immobilized antibodies to the antigen in human urine and optionally a second section with immobilized antibodies to the second antigen.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.